The research program described here aims at a full understanding of gene regulation by the steroid hormone ecdysone. Ecdysone regulates the development of insects and induces metamorphosis. Eip28/29, and ecdysone-responsive gene from Drosophila, is a model for steroid- regulated genes. Like other examples of such genes, its activity can be either elevated or depressed by the hormone, depending upon tissue and developmental stage. For example, Eip28/29 is induced by ecdysone in blood cells (and in some cell lines), but at the same time it is repressed by ecdysone in the epidermis and fat body. The regulatory DNA sequences (ecdysone response elements--EcREs--and other cis-acting elements) responsible for these diverse responses are distinct. The goal of the experiments proposed here is to understand ecdysone regulation in each class of tissue by identifying the relevant regulatory sequences, identifying and cloning the proteins that interact with them, and testing possible regulatory mechanisms. Mutant regulatory regions will be tested in cultured cells (by transient expression) and in the epidermis (by P element-mediated transformation of flies). Proteins which interact with these sequences will be identified by their binding to specific DNA sequences and/or by their activities in modifying transcription in vitro. Such proteins should include a variety of transcription factors, including factors which affect either EcRE-receptor binding or receptor activity after binding. They should also include transcription factors with more general functions. Since signal transduction by ecdysone is extremely similar to that of the vertebrate steroids, the results should provide useful insights into the activities of the more medically relevant vertebrate hormones. Similarly, transcription factors identified in Drosophila are likely to play important roles in gene regulation in other organisms.